The ubiquitin system of protein modification permeates every field in biology and is intimately linked to the most prevalent human diseases, including cancer, neurodegeneration, inflammation, and viral infections. With ~50 E2s and an estimated several hundred E3s, the ubiquitin system constitutes one of the most byzantine enzyme systems in eukaryotes. Whereas the basic principle of the E1-E2-E3 enzymatic cascade is now well established, the biological functions and regulation of ubiquitylation enzymes remain largely a mystery, mainly due to our inability to rapidly and comprehensively match ubiquitylation enzymes with their cellular substrates. As a solution to this predicament, which forestalls the entire ubiquitin field, a new activity-based protein capture and profiling technology, named SPASS (Solid Phase Assay for Ubiquitylation Substrate Screening) is proposed. SPASS simultaneously addresses three contemporary challenges: (1) It allows the unbiased identification of ubiquitylation substrates in an E2-specific manner. (2) Whereas structural information has proven unable to match the ~50 E2 with their hundreds of cooperating E3s, SPASS methodology provides this capability. (3) Lastly, the specific lysine residues that are modified are unknown for most substrates, a shortcoming that is equally addressed by the SPASS substrate capture and identification methodology. In the present project, SPASS will be applied to the profiling of ~15 E2s in a syngeneic tumor initiation and progression model of human prostate cancer. The main goal is to identify the substrates and cooperating E3 enzymes of these E2s, to pinpoint the substrate lysines that are modified, and to quantify differences in substrate utilization as prostate cells progress from an immortalized to a tumorigenic phenotype. The main deliverables will be a comprehensive platform and publicly available datasets that will provide cancer researchers and clinicians with a novel resource to link genomic and proteomic data with cancer progression. PUBLIC HEALTH RELEVANCE: The essential involvements of ubiquitylation enzymes (E2 and E3) in cellular regulation have placed them squarely at the center of prevalent human diseases, in particular cancer. Consequently, several pharmaceutical companies are already developing strategies for modulating ubiquitin system activity in a therapeutic context. A comprehensive understanding of the exact substrates and pathways controlled by ubiquitylation enzymes such as enabled by the studies proposed in this application will lay the foundation for harnessing the full potential of such efforts.